Surface-sensitive detection of hybridization at equilibrium

ABSTRACT

A method of analysing nucleic acid sequences comprises measuring by surface sensitive detection technique the binding interaction between a first nucleic acid sequence and a second nucleic acid sequence, one of the first and second nucleic acid sequences being immobilized to a solid phase surface, to determine the affinity or an affinity related parameter for the binding reaction as indicative of the extent of complementary between the first and second nucleic acid sequences. The method is characterized in that the measurement of the binding interaction is performed at annealing conditions adjusted such that the dissociation rate constant for the binding interaction corresponding to full complementarity between the first and second nucleic acid sequences is greater than about 10 -  per second, thereby permitting equilibrium for the interaction to be rapidly attained.

FIELD OF THE INVENTION

The present invention relates to an improved method of nucleic acidsequencing based on the detection and measurement of hybridizationinteractions.

BACKGROUND OF THE INVENTION

The generation of DNA sequence information has dramatically increaseddue to the large programs for sequencing of the human genome. Today thatwork is mainly done by traditional gel electrophoretic separation of DNAfragments terminated at different positions, either enzymatically(dideoxy chain termination method according to Sanger et al., Proc.Natl. Acad. Sci. USA 74: 5463-5467 (1977)) or chemically (chemicaldegradation method according to Maxam and Gilbert, Proc. Natl. Acad.Sci. USA 74: 560-564). These systems are, however, both time- andlabour-intensive.

There is therefore a general need for more effective methods for de novosequencing of DNA as well as for repeated sequencing of known sequencesfor analysis of mutations, such as point mutations. The mutationanalysis will increase as more information will be gathered about thecorrelation between different diseases and mutations and also due to theneed to verify deliberately introduced mutations in biotechnology work.

Sequencing by hybridization (SBH) (see e.g. Drmanac et al., Genomics 4:114; Strazoski et al., Proc. Natl. Acad. Sci. USA 88: 10089 (1991);Bains and Smith, J. Theoretical Biol. 135: 303 (1988); and U.S. Pat. No.5,202,231) has become an interesting alternative to traditionalsequencing with a potential for higher through-put of information. Thistype of system utilizes the information obtained from multiplehybridizations of the polynucleotide of interest, using shortoligonucleotides to determine the nucleic acid sequence. However, thereare several technical problems associated with this technology. Forexample, while today there are ways to build arrays of oligonucleotideson a chip based on the synthesis of oligoprobes and photolitographictechniques, it is still complicated to provide on a chip the large setof oligonucleotide probes required for determining a random nucleic acidsequence. Further, the detection of interaction of labelled target DNAis normally done by fluorescent or radioactivity measurements. Suchdetection is dependent on washing of the chip to get rid of residuallabelled target molecules and the oligoprobes must therefore bind ratherstrongly to the target molecules. There are also problems with thebinding of oligoprobes with a single base mismatch in combination withthe different sensitivity to washing conditions dependent on base paircomposition, G:C being more stable than A:T. One attempt to overcomesuch problems is to use tetraalkylammonium salts that eliminate thedifference in stability of G:C and A:T base pairs.

Even if differences in base composition can be compensated for, thewhole SBH procedure is based on interaction, washing, and detection ofhybridized target DNA and oligoprobe. The conditions for thehybridization thus have to be adjusted for a stable hybridization whichcan be detected only after several washing cycles. Dependent on theposition of the mismatch of single bases, base composition, oligoprobelength and temperature, there will be several hybridizations ofoligomers that will show up as weaker binding and such interactions willbe problematic to determine. Temperature and salt gradients elution havebeen suggested but are difficult to elaborate technically.

Due to the conditions needed for hybridization there is also always apotential risk for the target DNA to hybridize to itself due tocomplementary regions of the DNA.

A major disadvantage of SBH is, however, that the information isexclusively based on short-range information and the fact that overlapsare unique. Success is dependent on whether or not there are repeatedsequences in the nucleic acid to be analysed. The need and importance ofrepeated sequences are known from several situations, not least in theanalysis of genes like, for example, the gene for Huntington's diseasewhere repeated sequences and the amount of repeats have clinicalrelevance.

For the analysis of known sequences or of a particular site, mutationanalysis may be advantageous. An example of this is the mutationdependent tumour frequency found for proteins such as p53. Binding ofp53 to DNA is crucial for a correct control of cell growth and mutationdependent methods for therapy are likely to be developed. Furthermore,the kind of mutation detected may affect the treatment and aid inselecting the appropriate drug.

Label-free real-time measuring techniques, such as those based onsurface plasmon resonance (SPR), have been used to study thehybridization of DNA and oligomeric probes (oligoprobes). Attempts havealso been made to analyse the kinetic information of the hybridizationto determine the degree of hybridization, e.g. to detect mutantsequences, as described in Biosensor Application Note 306, 1994,Pharmacia Biosensor AB, Sweden. It has, however, been found that suchanalyses are difficult to use for obtaining relevant mismatchinformation as the kinetics for hybridization is complex under theconditions for hybridization normally used, which result in theformation of relatively stable hybridization complexes with longhalf-lives.

WO 93/25909 discloses the use of label-free techniques mentioned abovein combination with immobilised receptors, specifically antibodies,which are selected or designed to have a high dissociation constant, or"off-rate", for the binding of analyte to the receptor. Such a detectionsystem will rapidly respond to changes in the analyte concentration andregeneration of the sensing surface supporting the receptor will not berequired. Typically, the receptor is selected such that the dissociationrate constant (k_(off) or k_(diss)) for a particular analyte of interestis higher than 10⁻² per second.

WO 95/00665 discloses a method of providing the sequence of a singlestranded nucleic acid molecule, which when hybridized to a complementarysingle stranded molecule results in a double (duplex) structure having apreselected value for a free energy parameter, such as ligand binding,melting temperature or affinity for a target sequence. Thereby nucleicacid molecules may be produced which are tailored for specificapplications, e.g. nucleic acid molecules with a defined affinity for aligand which binds to the DNA and regulates the expression of a proteinencoded by the nucleic acid.

SUMMARY OF THE INVENTION

One object of the present invention is to provide a nucleic acidsequencing method based on hybridization interactions at a solid phasesurface, which method is more rapid to perform than the prior artmethods and which may not require regeneration of the surface betweenmeasurements.

Another object of the invention is to provide a sequencing method whichis more sensitive than the prior art methods to obtain mismatchinformation, i.e. in determining whether there is full complementaritybetween hybridizing nucleic acid fragments or not.

Still another object of the invention is to provide a sequencing methodwhich permits the use of short oligonucleotide probes.

Another object of the invention is to provide a sequencing method whichpermits the detection of repeated sequences.

Yet another object of the invention is to provide a sequencing methodwhich is less susceptible to the presence of complementary regions in atarget nucleic acid sequence.

It has now according to the present invention been found that the aboveand other objects and advantages may be attained by providing forreaction conditions which, on one hand, give lower affinity but, on theother hand, permit rapid association and dissociation in thehybridization interactions at the surface. More particularly, thereaction conditions should be selected such as to obtain a highdissociation rate constant for the hybridization event.

In contrast to the above-mentioned WO 93/25909 which describes a highdissociation rate obtained by selection or design of molecularproperties of the receptor, the present invention provides for thedesired high dissociation rate by appropriate adjustment of the reactionconditions.

The present invention thus relates to a method of analysing nucleic acidsequences, which method comprises measuring by surface sensitivedetection technique the binding interaction between a first nucleic acidsequence and a second nucleic acid sequence, one of the first and secondnucleic acid sequences being immobilized to a solid phase surface, todetermine the affinity or an affinity related parameter for the bindingreaction as indicative of the extent of complementarity between thefirst and second nucleic acid sequences. The invention is characterizedin that the measurement of the binding interaction is performed atannealing conditions adjusted such that the dissociation rate constantfor the binding interaction corresponding to full complementaritybetween the first and second nucleic acid sequences is greater thanabout 10⁻³ per second, thereby permitting equilibrium for theinteraction to be rapidly attained.

The term nucleic acid is to be interpreted broadly and in addition toDNA and RNA also includes nucleic acid analogues, such as modified DNAor RNA, or other hybridizing nucleic acid like molecules, such as PNA(peptide nucleic acid).

The term annealing as used herein refers to the combination andinteractions of two complementary nucleic acids.

The first and second nucleic acids may each be single-ordouble-stranded, or single-stranded DNA or RNA complexed with adifferent molecule, such as a modified nucleic acid, PNA, a peptide,etc.

In a preferred embodiment, the dissociation rate constant is higher thanabout 10⁻² per second, especially higher than about 10⁻¹ per second.

Other preferred embodiments of the invention are described below.

DETAILED DESCRIPTION OF THE INVENTION

The present invention thus relates to the analysis of the interactionbetween a first nucleic acid and a complementary second nucleic acidunder annealing conditions where high kinetic (dissociation rate)constants are present in the interaction and equilibrium of the reactiontherefore can be achieved rapidly, i.e. that the reaction will readilyreach equilibrium within a reasonable time during the experiment. Thisis valid for the formation of e.g. DNA--DNA dimers as well as sequencedependent formation of other complexes of nucleic acids.

The necessary conditions for the desired high dissociation rate may beobtained by proper selection of one or more of (i) the solvent, ionicstrength and/or pH, (ii) the reaction temperature and (iii) the nucleicacid probe length. In the second case (ii), which is presentlypreferred, a reaction temperature is selected which is closer to themelting temperature, Tm, in the annealing of complementary DNA fragmentsthan used in conventional hybridization assays for the particularchemical environment or above the melting temperature ("Tm" being thetemperature at which half of the hybridization is lost at equilibrium).The annealing of the nucleic acids reflects the response obtained for aparticular affinity at the conditions used and the relative affinity canbe determined with high precision.

Thus, for a certain interaction, the dissociation rate constant(k_(diss)) is dependent on the reaction temperature or the reactionmedium. While the equilibrium response level may be controlled byselection of nucleic acid concentration, the reaction kinetics will bedependent on the temperature and the reaction medium.

The method of the invention may be performed by analysing theinteraction of either (i) the target nucleic acid, such as a target DNA,over different oligomeric probes, such as oligoDNA probes, immobilizedon the solid phase surface, or in other words, sensing surface, or (ii)a series of oligomeric probes over a surface having a target nucleicacid immobilized thereon.

It is readily understood that under standardized conditions with knownsolution concentrations, a single determination for each oligoprobe andtarget molecule, respectively, may be sufficient for a comparison to astandard value. As is per se known in the art, by using a concentrationclose to the inverse of the affinity constant, the sensitivity tochanges in affinity will be at its maximum.

There are several advantages of working at annealing conditions closerto the melting temperature than that conventionally used forhybridization or above that, as will be explained below.

With reference first to method aspect (i) above with oligoprobesimmobilized on the sensing surface, this variant may be performedsequentially with a different single oligoprobe immobilized on thesensing surface or with an array of different oligoprobes immobilized.

With dissociation rate constants higher than about 10⁻³ s⁻¹ theequilibrium will be reached rapidly, and there may be no need forregeneration of the oligoprobe--supporting sensing surface as thecomplex formed at the surface will dissociate within a few minutesspontaneously. The affinity can thus rapidly be determined and a newsample of target nucleic acid can be analysed without regeneration ofthe sensing surface.

By using a series of oligoprobes, each representing a part of a knownsequence, mutations in the target DNA can be analysed as a decrease inaffinity for the particular oligoprobe interaction with the targetnucleic acid, and the position for the mutation may also be identified,as will be described in more detail below. If all possible combinationsof oligoprobes can be analysed, it is readily understood that not onlythe position for a mutation can be determined but also the base changein question.

Measurements of affinity of the interaction under such annealingconditions that provide for a high dissociation rate constant will alsoreduce problems associated with target nucleic acid intramolecularcomplementary structures, as the latter will have a lower tendency to beformed under rapid kinetic conditions and be under exchange with theimmobilized oligomeric nucleic acid on the surface.

Another problem usually experienced in sequencing by hybridization (SBH)is to obtain a good nucleic acid preparation and to know theconcentration of the amount of active nucleic acid, the term "active"referring to the hybridization capability. Under conditions ofcontinuous flow and a diffusion rate limited association, the initialassociation of target DNA to an immobilized oligoprobe will beconcentration dependent and can be used for concentration determinationof the active target DNA. Such a concentration determination will berequired for correlating the affinity measurements to literature data.

The well-known prior art problem in sequencing by hybridization relatedto the presence of repeated sequences can also be overcome by thepresent procedure of affinity measurements under annealing conditionswith high dissociation rate constants. Since a repeated sequence caninteract with different oligoprobes immobilized on the sensing surface,the avidity effects of such multiple interactions will give rise to ahigher "apparent" affinity than expected and a much smaller "apparent"dissociation rate constant. The affinity determinations will thus besensitive for repeated sequences as an increase in apparent affinity,whereas a decrease in affinity will be correlated to a mismatch.

The above described first method aspect of the invention, where anoligoprobe is immobilized on the sensing surface and a larger nucleicacid fragment to be sequence analysed is present in solution, issuitable for large arrays of oligoprobes and a detection system that cananalyse a large amount of interaction at the same time. However, thereversed analytical situation according to the above-mentioned secondmethod aspect relating to the sequential analysis of oligoprobeinteractions with immobilized target nucleic acid is also useful.

Under such conditions, for example, the amount of repeated sequences canbe correlated to the amount of bound oligoprobes as long as they reactindependently of each other. In this way repeated sequences can bequantified and will not be dependent on avidity effects as mentionedabove.

The increase in binding due to several binding positions on theimmobilized target nucleic acid can therefore be described with the samedissociation rate constant as the single interaction, in contrast to thecase of a multiple interaction with target nucleic acid in solutionwhere the interactions with several different oligoprobes immobilized onthe surface will give rise to an increased signal coupled to a lowerdissociation rate constant.

Since in the present invention, association rate is used to measure theconcentration of the analyte, the equilibrium response level will be ameasure of affinity, a complete match of hybridizing sequences having ahigher afffinity than a mismatch. A higher equilibrium level with thesame dissociation rate constant will therefore be a measure of theamount of repeated sequences, whereas an increase in signal incombination with a smaller "apparent" dissociation rate constant will bea measure of multiple interaction for that sequence. Information fromthe dissociation part of the interaction can thus be used in combinationwith the affinity measurement for verification of the interaction and tomake the method as sensitive as possible for detecting a mismatch.

To identify positions for a mutation in a nucleic acid fragment, such asa DNA fragment, it may be advantageous to use a scanning approach. Ifthe mutation position is unknown, an exemplary procedure may be asfollows:

The DNA fragment is immobilized on the sensing surface.

The position for the mutation is then scanned by sequentially contactingthe surface with solutions of oligoprobes, each covering a respectivepart of the whole DNA target molecule.

The part or parts of the DNA target which are found to contain amutation are then subjected to a detailed analysis of the position ofthe mutation by overlapping probe analysis in a "minisequencing" format.

The identity of the base mutation may then be determined by testing thefour base variants (A, C, T and G) of an oligoprobe with respect to themutated position. If desired, this last step can be done directly in theabove minisequencing format if all possible combinations of oligoprobesare tested.

The above described procedure may be beneficial as sequencing analysesoften will be done with samples which do not contain any mutationstherein and it is understood that the described scanning approach can bea very rapid procedure.

A corresponding scanning procedure may, of course, be performed in thereversed mode with the oligoprobes immobilized on the surface and thetarget nucleic acid in solution.

The surface sensitive detection used in the method of the invention maybe obtained by various detection systems. In a suitable type ofdetection system, a change in a property of the sensing structure ismeasured as being indicative of binding interaction at the sensingsurface. Among these methods are, for example, mass detecting methods,such as piezoelectric, optical, thermo-optical and surface acoustic wave(SAW) methods, and electrochemical methods, such as potentiometric,conductometric, amperometric and capacitance methods. It is alsopossible to use short range radioactivity, such as scintillationplastics in close proximity to the interaction position of ³ H or othershort range ionising radiation.

Among optical methods may particularly be mentioned those that detectsurface refractive index, such as reflection-optical methods, includingboth internal and external reflection methods, e.g. ellipsometry andevanescent wave spectroscopy (EWS), the latter including surface plasmonresonance spectroscopy (SPRS), Brewster angle refractometry, criticalangle refractometry, frustrated total reflection (FTR), evanescent waveellipsometry, scattered total internal reflection (STIR), optical waveguide sensors, evanescent wave based imaging, such as critical angleresolved imaging, Brewster angle resolved imaging, SPR angle resolvedimaging, etc., as well as methods based on evanescent fluorescence(TIRF) and phosphorescence.

Among the optical methods mentioned above, especially SPRS has attractedmuch attention recently. The phenomenon of SPR is well known. In brief,SPR is observed as a dip in intensity of light reflected at a specificangle from the interface between an optically transparent material, e.g.glass, and a thin metal film, usually silver or gold, and depends onamong other factors the refractive index of the medium (e.g. a samplesolution) close to the metal surface. A change of refractive index atthe metal surface, such as by the adsorption or binding of materialthereto, will cause a corresponding shift in the angle at which SPRoccurs. To couple the light to the interface such that SPR arises, twoalternative arrangements may be used, either a metallized diffractiongrating (Wood's effect), or a metallized glass prism or a prism inoptical contact with a metallized glass substrate (Kretschmann effect).For further details on SPR, reference is made to our WO 90/05295.

The measurements in the present invention are preferably performed in aflow system type biosensor, permitting rapid kinetics to be identified.An example of such a biosensor system is the BIAcore® system (marketedby Pharmacia Biosensor AB, Uppsala, Sweden).

The present invention will now be illustrated by the followingnon-limiting Examples, reference also being made to the accompanyingdrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an overlay plot of sensorgrams (response vs time) for thehybridization of matching as well as mismatching 7- and 8-mers(NIPE32-36) to an immobilized target DNA sequence (BP22).

FIG. 2 is a plot of binding curves (equilibrium responses vsconcentration) for different concentrations of the oligoprobes in thehybridization in FIG. 1.

FIG. 3 is a plot of binding curves for a number of oligoprobes (BP35,47-56) to an immobilized p53 derived wild-type (wt) target DNA sequence(BP44).

FIG. 4 is another type of plot of the binding curves in FIG. 3.

FIG. 5 is a corresponding plot to that in FIG. 4 showing the binding ofthe same oligomers (BP35, 47-56) to a mutant target DNA sequence (BP45)

FIG. 6 is a corresponding plot to that in FIG. 5 showing the binding ofthe same oligomers (BP35, 47-56) to another mutant target DNA sequence(BP46).

FIG. 7 is a resume of the binding curves in FIGS. 4 to 6.

FIG. 8 is an overlay plot of sensorgrams for the binding of a wt targetDNA (BP68) to a set of immobilized oligoprobes (BP63, 64 and 66).

FIG. 9 is an overlay plot of sensorgrams for the binding of a mutanttarget DNA (BP69) to a set of immobilized oligoprobes (BP63, 64 and 66).

FIG. 10 is an overlay sensorgram showing the sequential hybridization ofa series of oligoprobes (BP47-51, BP35, BP52-56) to a target wild-type(wt) DNA (BP44) and mutant DNA (BP45) in series.

FIG. 11 is a diagram showing a subtraction of the two sensorgrams forthe wt and mutant target DNA in FIG. 10 from each other.

FIG. 12 is a plot of binding curves (equilibrium response vsconcentration) for the hybridization of an oligoprobe (BP61) to animmobilized target DNA (BP46) at different temperatures.

EXAMPLES

The experimental work described below is essentially based on thehybridisation of oligonucleotide probes having matching 8-mer sequences.The target oligonucleotide was in no case exceeding the size of a50-mer. A set of reagents with oligosequences derived from exon 6 of thep53 gene was used. Hybridisations were performed in two formats, eitherstraight, i.e. the probe (the 8-mer) in solution and the target DNA onthe surface, or reversed, with the probe on the surface and the targetin solution.

MATERIALS AND METHODS

The analytical instrument used for the analyses was either a BIAcore® ora BIAcore ® 2000 system (Pharmacia Biosensor AB, Uppsala, Sweden), whichare biosensor systems based on SPR and having four flow channels. Assensing surface was used Sensor Chip CM5 or SA5, both with instrumentimmobilised streptavidin (SA), or D×500 or D×40 matrices with batchimmobilised SA.

Generally, a buffer consisting of 10 mM Hepes, 0.5 M NaCl, 3.4 mM EDTA,0.002% Surfactant P20, pH 7.4 was used for hybridisation. The samebuffer was used for capturing of biotinylated ligands. The ligandconcentration used for capturing was ˜1 μg/ml. Variable contact timeswere used to control ligand surface concentrations. Oligonucleotides,denoted "BP" or "NIPE", were purchased from Pharmacia Biotech AB,Uppsala, Sweden (BP1-69), Scandinavian Gene Synthesis, Koping, Sweden(NIPE8, 19-22) or KEBO Lab, Stockholm, Sweden (NIPE32-36).

When necessary, HCl, pH 2.15, was used for the regeneration of thesurface between each hybridisation cycle. The hybridisation temperaturewas 25° C. The equilibrium response ("R_(eq) ") was obtained by takingthe report point at steady-state in an active flow cell, subtracted bythe corresponding value in a flow cell without captured oligonucleotide.The R_(eq) -response for each probe concentration was exported to theBIAeval™ software (Pharmacia Biosensor AB) and the affinity wasdetermined by a non-linear procedure. A contact time of three minutesand a flow rate of 15 μl/min was used for a typical cycle of analysis.The hybridisation studies were performed either (i) with a biotinylatedtarget on the surface and 8-mer probes in solution or (ii) withbiotinylated probe on the surface and the target molecule in solution.These procedures are referred to as "straight" and "reversed" mode,respectively.

Example 1 Hybridization of Different Oligoprobes to Immobilized TargetDNA Under Equilibrium Conditions

The following oligonucleotides were tested:

NIPE32: 3' CGTAGAAT (fully matched)

NIPE33: 3' G-------

NIPE34: 3' ₋₋ -------

NIPE35: 3' -------G

NIPE36: 3' -------₋₋

"-" indicates the same base as the fully matched sequence, and "₋₋ "indicates no base.

The following target DNA sequence was used: BP22: 5' BioTTTTTTTTGCATCTTATTTTTTTT SEQ ID NO: 1, where "Bio" indicates a biotinlabel.

Hereinafter, "RU" refers to resonance units, and "Fc1" refers to flowchannel 1, etc.

BP22 was captured (1523 RU) in Fc1 on a SA5 chip and 0.4 μM probe washybridised. The results are shown in FIG. 1. As appears from the Figure,the 3'-end mismatch does not bind, while the 5'- dito binds, but not asstrongly as the matched 8-mer. Of the 7-mers, NIPE36 binds, but notNIPE34, which might reflect the different GC-contents, 3/7 and 2/7,respectively. R_(eq) -responses were taken as the flow cell differenceFc1-Fc2.

BP22 was then captured on SA5 at different surface concentrations: Fc1,2, 3 & 4 with 296, 808, 1314 and 0 RU, respectively. Then, NIPE 32-36were passed serially over the chip at concentrations of 15 nM-32 μM. InFIG. 2, all probes (NIPE32-36) are compared with R_(eq) -responsesplotted vs. concentration. As can be seen in the Figure, NIPE32, 35 and36 reach R_(max) at a high concentration and it is possible to determinethe affinities. The equilibrium dissociation constant (K_(D)) was 0.85,1.5 and 1.5 μM, respectively. The 3'-end mismatch (NIPE33) and the 7-mer(NIPE34) had much lower affinities. As seen from the Figure, the R_(max)value for NIPE35 is ˜15% lower than for NIPE32. This is probably aneffect of loss of ligand or sub-optimal regeneration with each cycle.The baseline drift was on average 0.7 RU/cycle over 100 cycles and theprobes were run in order starting with NIPE32 and ending with NIPE36.With 1300 RU of a 24-mer ligand, 430 RU of an 8-mer probe would beexpected to bind. At R_(max), 380 RU were bound, e.g. a near 90%occupancy. When NIPE35 comes in, after 60 cycles, ligand concentrationmay be decreased, but also, some previously hybridised probe may havegot stuck on the target, thus decreasing the active surfaceconcentration. The lower response of NIPE36 is probably an effect of thelower molecular weight.

A second set of probes was then constructed with 8-mer oligonucleotides,covering the whole target molecule with one base shift per probe. Threebiotinylated target molecules based on the p53 system were alsoconstructed, one "wild-type" to which all probes were fully matched andtwo targets with either a substitution of C for T or T for A.

    5'-B-tttCCTCAGCATCTTATCCGAGttt                                                                    BP44 (SEQ ID NO:2)                                                                          "wt"                                           - 5'-B-tttCCTCAGCATTTTATCCGAGttt BP45 (SEQ ID NO:3) "mutC>T"                  - 5'-B-tttCCTCAGCAACTTATCCGAGttt BP46 (SEQ ID NO:4) "mutT>A"                  -         GGAGTCGT BP47 (SEQ ID NO:5)                                         -          GAGTCGTA BP48 (SEQ ID NO:6)                                        -           AGTCGTAG BP49 (SEQ ID NO:7)                                       -            GTCGTAGA BP50 (SEQ ID NO:8)                                      -             TCGTAGAA BP51 (SEQ ID NO:9)                                     -              CGTAGAAT BP35 (SEQ ID NO:10)                                   -               GTAGAATA BP52 (SEQ ID NO:11)                                  -                TAGAATAG BP53 (SEQ ID NO:12)                                 -                 AGAATAGG BP54 (SEQ ID NO:13)                                -                  GAATAGGC BP55 (SEQ ID NO:14)                               -                   AATAGGCT BP56 (SEQ ID NO:15)                              -             TCGTAGAT BP57 (SEQ ID NO:16)                                    -             ACGTAGAA BP58 (SEQ ID NO:17)                                    -                  GAATAGGG BP59 (SEQ ID NO:18)                               -                  CAATAGGC BP60 (SEQ ID NO:19)                               -              CGTTGAAT BP61 (SEQ ID NO:20)                                   -              CGTAAAAT BP62 (SEQ ID NO:21)                            

BP57 and 58 have A or T in either end and should be compared with BP51.Correspondingly, BP59 and BP60 have a G or C in either end and should becompared with BP55. BP61 and 62 are fully matched to the "mutants" BP46and BP45, respectively.

In the first experiment, the binding of probes BP35 and BP47-56 to thetarget BP44 was tested. The concentration range was 31 nM-64 μM and thebinding curves obtained are presented in FIG. 3. A striking observationis that the range of affinity is quite wide for these 8-mer probesspanning from 2×10⁵ -6×10⁶ M⁻¹. A fairly good correlation between theaffinities obtained and Tm-values calculated as 2×n(A+T)+4×n(G+C) wasfound. FIG. 4 shows the binding curves of the probes on the "wt"-target.By comparing this figure to FIG. 5, which shows corresponding bindingcurves on a mutant target (BP45), where C was changed to T, the effectof single base pair mismatch is clearly demonstrated. The position ofthe mismatch has a dramatic effect on binding. The same effect is seenin FIG. 6 with target BP46, where T was changed to A. FIG. 7 is a resumeof FIGS. 4-6, showing only the R_(eq) -responses of the highestconcentrations of each probe. The "U-shaped profiles" of the mutanttargets differ clearly from the profile of the wt-target, suggestingthat it is possible to discriminate perfectly matched probes from endmismatched ones. Comparing the highest response for each probe was foundto be discriminative.

Example 2 Hybridization of Different Target DNA Sequences to ImmobilizedOligoprobes Under Equilibrium Conditions

A new set of modified oligonucleotides based on those described abovewas designed. Three biotinylated probes containing an 8-mercomplementary sequence with a spacer tri-T in the 5'-end wereconstructed. Two 19-mer targets, one "wild-type" and one "mutant" with Cchanged to T, were also constructed.

    5'-CCTCAGCATCTTATCCGAG                                                                          BP68 (SEQ ID NO:22)                                                                        "wt"                                              - 5' ---------T---------- BP69 (SEQ ID NO:23) "mutC>T"                        -         GTAGAATAttt-B-5' BP63 (SEQ ID NO:24)                                -     AGTCGTAGttt-B-5' BP64 (SEQ ID NO:25)                                    -            GAATAGGCttt-B-5' BP66 (SEQ ID NO:26).                     

BP68 and 69 correspond to BP44 and 45, except that they are notbiotinylated and the extra tri-T ("ttt") of either end has been deleted.BP63, 64 and 66 correspond to BP52, 49 and 55, respectively.

BP63, 64 and 66 were captured in Fc1-3 (554, 782 and 651 RU,respectively) and BP68 and 69 were passed over the flow cells in aserial mode. The concentrations used were 63 nM-64 μM. The results areshown in the sensorgrams in FIG. 8 (BP68) and FIG. 9 (BP69), where thebinding of the two targets to the three probes are compared (blanksurface sensorgram is subtracted). As seen in the Figures, the wt targetbinds to all probes, but with different affinities. BP64 binds best,then BP63 and finally BP66 with the lowest affinity. The mutant targetshows reduced binding to all three probes, with no binding at all toBP63. This is the probe with the central mismatch. Thus, the 5'- and3'-end mismatched probes still bind the target, but with about half theR_(eq) -response of the wt target. However, the binding deviatessligthly from that obtained in the straight mode; the on-rate isslightly lower and the binding looks more stable. At the lowerconcentrations, no steady-state is attained, but a slow binding isobserved. The R_(eq) -responses were used to estimate the affinities viaBIAeval™. In Table 1 below, the values obtained are demonstrated.

                  TABLE 1                                                         ______________________________________                                        Target      Probe      Mismatch K.sub.A (M.sup.-1)                            ______________________________________                                        BP68 (wt)   BP63       no       1.5 × 10.sup.5                            BP68 (wt) BP64 no 3.2 × 10.sup.5                                        BP68 (wt) BP66 no 3.1 × 10.sup.5                                        BP69 (mutant) BP63 internal --                                                BP69 (mutant) BP64 5'-end 0.5 × 10.sup.5                                BP69 (mutant) BP66 3'-end 0.3 × 10.sup.5                              ______________________________________                                    

Thus, also the reversed mode shows a clear discrepancy between endmismatch and internal mismatch. Also in this experiment, 5'-end mismatchhas less effect than 3'- dito with respect to affinity.

As demonstrated above, an analysis of oligonucleotide hybridisation canbe performed by measuring annealing under equilibrium conditions. Themeasurements are performed near or above the Tm-values of the 8-merprobes, which allows a very sensitive analysis. This is a majoradvantage compared to many other methods, where stringency conditionsmust be very carefully selected if precision should not be sacrificed.The analysis is fast, less than 3 minutes contact time, about 5min/cycle, and regeneration is not needed. Fully matched 8-mer probesbind with μM-order affinity (25° C., 0.5 M salt).

Example 3

A series of oligoprobes BP47-BP51, BP35 and BP52-56 were introduced intoBIAcore® 2000 with immobilized target wild-type (wt) BP44 and mutantvariant BP45 on different surfaces passed in series by the liquid flow.The experiment was run at 30° C. at 32 μM concentration of the differentoligoprobes diluted in buffer. The flow rate was 15 μl/min and theinjected sample volume was 30 μl. The results are shown in FIG. 10.

As seen in the Figure, the dilution gave rise to a dilution effectduring the sample introduction and a rapid decrease in response signalduring the sample pulse. The diamond (♦) labelled curve (thick line)shows the interaction with target BP45 mutant and the thin line showsthe interaction with wt BP44. During the washing phase between theintroduction of the respective oligoprobes BP47-51, a dissociationsignal can be seen in the wt response curve, whereas the dissociationfor the other oligoprobes was rapid at the temperature used and cannotbe resolved from the change in buffer composition. As is clearlydemonstrated in the Figure, the response levels for wt and mutant weredifferent over the experiment where oligoprobes passes over the mutantposition.

FIG. 11 shows a subtraction of the two sensorgrams in FIG. 10 from eachother, demonstrating that the difference in response under the annealingconditions gave rise to an increase in signal at the position of amutation in the mini-sequencing type procedure performed. The diagramshows that the mutation starts with BP49 and ends with BP55 where thedifference in signal is well separated from the baseline. (The spikes inthe diagram are due to the short time differences for the sample to passover the two different surfaces with wt and mutant target DNA,respectively.)

Example 4

The response at equilibrium for the interaction of oligoprobe BP 61 infull match with immobilized target BP 46 was studied at differentconcentrations and different temperatures ranging from 5 to 35° C. Theexperiment was run in buffer 2×SSC, a citrate buffer with 0.3 M NaCl, pHabout 7. The flow rate was 15 μl/min and the injected sample volume was45 μl. The results are shown in FIG. 12.

The affinity was calculated in the same way as in Example 1, and Table 2below gives the values for both the obtained equilibrium associationaffinity constant (K_(A)) and the corresponding dissociation rateconstant (k_(diss)) for the studied temperatures.

                  TABLE 2                                                         ______________________________________                                        Temp. (° C.)                                                                          K.sub.A  k.sub.diss                                            ______________________________________                                         5             1.51 × 10.sup.6                                                                  2.49 × 10.sup.-3                                  15 9.31 × 10.sup.5 0.02                                                 20 6.74 × 10.sup.5 0.11                                                 22 5.13 × 10.sup.5 0.14                                                 25 2.83 × 10.sup.5 >0.2                                                 28 2.01 × 10.sup.5 >0.2                                                 35 3.74 × 10.sup.4 >0.2                                               ______________________________________                                    

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 26                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - TTTTTTTTGC ATCTTATTTT TTTT          - #                  - #                    24                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - TTTCCTCAGC ATCTTATCCG AGTTT          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - TTTCCTCAGC ATTTTATCCG AGTTT          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - TTTCCTCAGC AACTTATCCG AGTTT          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - TTTGGAGTCG TACTTATCCG AGTTT          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - TTTCGAGTCG TACTTATCCG AGTTT          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - TTTCCAGTCG TAGTTATCCG AGTTT          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - TTTCCTGTCG TAGATATCCG AGTTT          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - TTTCCTCTCG TAGAAATCCG AGTTT          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - TTTCCTCACG TAGAATTCCG AGTTT          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                              - - TTTCCTCAGG TAGAATACCG AGTTT          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                              - - TTTCCTCAGC TAGAATAGCG AGTTT          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                              - - TTTCCTCAGC AAGAATAGGG AGTTT          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                              - - TTTCTTCAGC AAGAATAGGC AGTTT          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                              - - TTTCTTCAGC AACAATAGGC TGTTT          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:16:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                              - - TTTCCTCTCG TAGATATCCG AGTTT          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:17:                                    - 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-     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                              - - TTTCCTCAGC AAGAATAGGG AGTTT          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:19:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                              - - TTTCCTCAGC AACAATAGGC AGTTT          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:20:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                              - - TTTCCTCACG TTGAATTCCG AGTTT          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:21:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                              - - TTTCCTCACG TAAAATTCCG AGTTT          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:22:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                              - - CCTCAGCATC TTATCCGAG             - #                  - #                      - # 19                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:23:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                              - - CCTCATCATT TTATCCGAG             - #                  - #                      - # 19                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:24:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                              - - GTAGAATATT T               - #                  - #                      - #       11                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:25:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                              - - AGTCGTAGTT T               - #                  - #                      - #       11                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:26:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                              - - GAATAGGCTT T               - #                  - #                      - #       11                                                                 __________________________________________________________________________

We claim:
 1. A method of analyzing nucleic acid sequences, which methodcomprises measuring by surface sensitive detection technique the bindinginteraction between a first nucleic acid sequence and a second nucleicacid sequence, one of the first and second nucleic acid sequences beingimmobilized to a solid phase surface, to determine the affinity or anaffinity related parameter for the binding reaction as indicative of theextent of complementarity between the first and second nucleic acidsequences, wherein the measurement of the binding interaction isperformed at annealing conditions adjusted such that the dissociationrate constant for the binding interaction corresponding to fullcomplementarity between the first and second nucleic acid sequences isgreater than 10⁻³ per second, thereby permitting equilibrium for theinteraction to be rapidly attained.
 2. The method according to claim 1,wherein said measurement is performed at equilibrium.
 3. The methodaccording to claim 1, wherein the dissociation rate constant is greaterthan 10⁻² per second.
 4. The method according to claim 1, wherein thefirst nucleic acid sequence is at least one oligonucleotide probe andthe second nucleic acid sequence is at least one target nucleic acidsequence to be sequence analyzed.
 5. The method according to claim 1,wherein the first nucleic acid and second nucleic acid sequencesindependently are single- or double-stranded DNA or RNA orsingle-stranded DNA or RNA complexed with a different molecule whereinthe different molecule is a molecule other than the single-stranded DNAor RNA.
 6. The method according to claim 1, wherein said adjustment ofthe annealing conditions comprises adjusting the temperature and/or thereaction medium.
 7. The method according to claim 4, wherein saidoligonucleotide probe or probes are bound to the solid phase surface. 8.The method according to claim 7, wherein the method comprises theadditional step of analyzing the dissociation constant for the bindingreaction to determine therefrom the possible presence of repeatedsequences in the target nucleic acid sequence.
 9. The method accordingto claim 4, wherein the target nucleic acid sequence is bound to thesolid phase surface.
 10. The method according to claim 9, wherein themethod comprises scanning the target nucleic acid sequence for amutation or mutations with a first set of oligonucleotide probes todetect a mutated part or parts of the target sequence, and then testinga detected mutated part or parts of the target sequence with a secondset of oligonucleotides to determine the position of the mutation ormutations and optionally the corresponding base change.
 11. The methodaccording to claim 1, wherein said first or second nucleic acidsequences are immobilized in an array at defined positions on the solidphase surface.
 12. The method according to claim 1, wherein the methodcomprises the additional step of determining the concentration of thefirst or second nucleic acid sequence at diffusion rate limitedconditions.
 13. The method according to claim 1, wherein said surfacesensitive detection technique is based on evanescent wave sensing. 14.The method according to claim 1, wherein the measurement is performed ina flow cell.